For the last 25 years, 2-D PAGE (two-dimensional polyacrylamide gel electrophoresis) has been the technique of choice for analysing the protein composition of a given cell type and for monitoring changes in gene activity through the quantitative and qualitative analysis of the thousands of proteins that orchestrate various cellular functions. Despite its extraordinary resolving power, 2-D PAGE has never been adopted for high throughput screening studies. This is because the first and second dimension gels are run separately, thus requiring two different running devices and making automation difficult. In addition, a high level of operator skill and knowledge is required to successfully complete a 2-D gel.
To achieve truly high throughput and reproducibility of 2-D gels it will be necessary remove as much of the operator intervention as possible. This could be accomplished by combining the first and second dimension gels in a single cassette and thus removing the need for the operator to interface the gels manually. This manual interfacing step is slow and cumbersome and a source of inter-operator differences thus leading to a lack of reproducibility. Another advantage of combining the two gels in a single cassette is the reduction of the running hardware to a single apparatus, thus increasing the possibilities of automation. Placing the gels together in a single cassette requires the ability to place the gels within a few millimetres of each other while maintaining the separation between the gels until the transfer of protein from the first to the second dimension is required. There are serious technical difficulties associated with combining the first and second dimension gels in a single cassette. Firstly, the solutions present, and thus the ionic conditions, in the two gels are quite dissimilar. The first dimension, isoelectric focusing, gel has very low ionic strength and is not compatible with high levels of salts or buffers. Isoelectric focusing is performed at high voltage, up to 10,000 volts, and very low current, typically below 1 mA per gel. Conversely the second dimension gel has a high concentration of buffer salts, usually in the 100 to 500 mM range, and the electrophoresis is performed at high currents in the range of 5 to 100 mA per gel. Thus, when placing these two gels in close proximity care must be taken to ensure that there is no contamination of the first dimension gel with buffer from the second dimension gel, which would result in very high current and subsequent burning of the first dimension gel.
The second problem that one faces is the loading of the sample onto the first dimension gel without allowing any of the sample to prematurely transfer to the second dimension gel. The best way of performing isoelectric focusing is to use immobilised pH gradients (IPG), which are typically supplied as dry strips and rehydrated with the sample solution, thus distributing the sample over the entire gel, which allows high protein loads. When the two gels are combined in a single cassette it is difficult to wet the entire surface of the IPG with sample without allowing some of the sample to transfer to the second dimension gel.
Attempts have been made to provide combined strip gels and slab gels for carrying out 2-dimensional electrophoresis. U.S. Pat. No. 4,874,490 discloses such an apparatus. However the proposed method is unwieldy and impractical and no examples of the method being carried out and actually working are given. With the recent upsurge in interest in the field of proteomics it becomes necessary to carry out many more separations than have previously been done. Separations, particularly two-dimensional separations are labour intensive and the only practical way of increasing the productivity of workers in the field is to provide methods and systems which are susceptible to automation. The method shown in U.S. Pat. No. 4,874,490, if it works at all, would be difficult, if not impossible, to mechanise.
U.S. Pat. No. 5,773,645 discloses a further apparatus for carrying out a two-dimensional separation on a common support. Again this specification does not disclose any examples or results from using the apparatus. Further the system disclosed would again be difficult and expensive to automate.
One object of the present invention is to provide a new method which overcomes the technical difficulties associated with combining and running the first and second dimension gels in a single cassette in an manner which may allow the method and system to be automated.
Any discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is solely for the purpose of providing a context for the present invention. It is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present invention as it existed in Australia before the priority date of each claim of this application.